Enzymatically-cleavable chemiluminescent fused polycyclic ring-containing 1,2-dioxetanes

• US 4,952,707 A
• Filed: 06/30/1988
• Issued: 08/28/1990
• Est. Priority Date: 06/30/1988
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Tropix Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Laird, Alison A., Edwards, Brooks, Voyta, John C., Bronstein, Irena Y.
• Primary Examiner(s): Raymond, Richard L.
• Application Number:
  US07/213,672
• Time in Patent Office: 789 Days
• Field of Search: 549/221, 549/510, 549/511, 549/332, 549/17, 549/43, 549/58, 549/223, 549/225, 549/226, 549/227, 549/359, 549/378, 549/388, 549/395, 549/462, 536/4.1, 536/18.1, 544/6, 544/35, 544/57, 544/70, 544/102, 544/230, 544/244, 544/265, 546/15, 546/23, 546/88, 546/102, 546/108, 546/121, 546/127, 546/139, 546/152, 546/270, 548/113, 548/119, 548/125, 548/147, 548/152, 548/216, 548/217, 548/260, 548/327, 548/336
• US Class Current: 549/221
• CPC Class Codes: C07C 2603/00 Systems containing at least three condensed rings
• CPC Class Codes: C07C 2603/74 Adamantanes
• CPC Class Codes: C07C 43/215 having unsaturation outside the six-membered aromatic rings
• CPC Class Codes: C07C 43/225 containing halogen
• CPC Class Codes: C07C 43/23 containing hydroxy or O-metal groups
• CPC Class Codes: C07C 49/84 containing ether groups, groups, groups, or groups
• CPC Class Codes: C07C 69/00 Esters of carboxylic acids; Esters of carbonic or haloformic acids ortho esters, see the relevant groups, e.g. C07C43/32
• CPC Class Codes: C07D 321/00 Heterocyclic compounds containing rings having two oxygen atoms as the only ring hetero atoms, not provided for by groups C07D317/00 - C07D319/00
• CPC Class Codes: C07F 9/65512 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte

Abstract

Chemiluminescent 1,2-dioxetane compounds are disclosed in which the molecule is stabilized at the 3-position on the dioxetane ring against decomposition prior to the molecule'"'"'s coming in contact with a labile group-removing substance (e.g., an enzyme that will cleave the labile group to cause the molecule to decompose to form at least one light-emitting fluorophore) and substituted at the 4-position on the dioxetane ring with a fused polycyclic ring-containing fluorophore moiety bearing a labile ring substituent whose point of attachment to the fused polycyclic ring, in relation to this ring'"'"'s point(s) of attachment to the dioxetane ring, is such that the total number of ring atoms separating these points of attachment, including the ring atoms at the points of attachment, is an odd whole number. These odd pattern substituted compounds decompose to emit light of greater intensity and of a different wavelength than that emitted by the corresponding even pattern substituted isomers. They are useful in detecting the presence or determining the concentration of chemical or biological substances in immunoassays, chemical assays and nucleic acid probe assays, and in chemical/physical probe procedures for studying the microstructures of macromolecules. Two or more of them can also be used in combination, or one or more of them can be used together with other chemiluminescent compounds, in multi-channel assays to detect two or more different analytes simultaneously. Novel intermediates used in the preparation of these odd pattern substituted compounds are also disclosed.

Claims

1. An enzymatically cleavable chemiluminescent 1,2-dioxetane compound having the formula:

##STR9## wherein R₁ is hydrogen, or a bond when R₂ is a substituent bound to the dioxetane ring through a spiro linkage, or an organic substituent that does not interfere with the production of light but satisfies the valence of the dioxetane ring carbon atom to which it is attached;

R₂ is a fused polycyclic ring-containing fluorophore moiety bound to the dioxetane ring through a single bond or a spiro linkage and having an enzymatically cleavable labile ring substituent containing a bond which, when cleaved, renders the polycyclic moiety electron-rich to in turn render the dioxetane compound decompasable to emit light, the enzymatically cleavable labile ring substituent'"'"'s point of attachment to the fused polycyclic moiety, in relation to the fused polycyclic moiety'"'"'s point of attachment to the dioxetane ring, being such that the total number of ring atoms separating these points of attachment, including the ring atoms at these points of attachment, is an odd whole number; and

T is a stabilizing group that prevents the dioxetane compound from decomposing before the enzymatically cleavable labile ring substituent'"'"'s bond is cleaved.

2. An enzymatically cleavable chemiluminescent 1,2-dioxetane compound as recited in claim 1 in which the odd whole number is 5 or greater.

3. An enzymatically cleavable chemiluminescent 1,2-dioxetane compound as recited in claim 2 in which the fused polycyclic moiety is the residue of a fused polycyclic aromatic hydrocarbon ring fluorophoric compound containing from 9 to about 30 ring carbon atoms, inclusive.

4. An enzymatically cleavable chemiluminescent 1,2-dioxetane compound as recited in claim 3 in which the residue of a fused polycyclic aromatic hydrocarbon ring fluorophoric compound is a naphthalene residue.

5. An enzymatically cleavable chemiluminescent 1,2-dioxetane compound as recited in claim 3 in which the residue of a fused polycyclic aromatic hydrocarbon ring fluorophoric compound is an anthracene residue.

6. An enzymatically cleavable chemiulminescent 1,2-dioxetane compound as recited in claim 2 in which the fused polycyclic moiety is the residue of a less than fully aromatic fused polycyclic hydrocarbon ring fluorophoric compound containing from 10 to about 30 carbon atoms, inclusive.

7. An enzymatically cleavable chemiluminescent 1,2-dioxetane compound as recited in claim 6 in which the residue of a less than fully aromatic fused polycyclic hydrocarbon ring fluorophoric compound is a fluorene residue.

8. An enzymatically cleavable chemiluminescent 1,2-dioxetane compound as recited in claim 2 in which the fused polycyclic moiety is the residue of a fused polycyclic heterocyclic ring fluorophoric compound containing from 9 to about 30 ring atoms, inclusive.

9. An enzymatically cleavable chemiluminescent 1,2-dioxetane compound as recited in claim 8 in which the residue of a fused polycyclic heterocyclic ring fluorophoric compound is a quinoline residue.

10. An enzymatically cleavable chemiluminescent 1,2-dioxetane compound as recited in any one of claims 3-9, inclusive, in which R₁ is a methoxy group, R₂ is bound to the dioxetane ring through a single bond and T is an adamant-2-ylidene group.

11. An enzymatically cleavable 1,2-dioxetane compound as recited in claim 10 in which the enzymatically cleavable labile ring substituent is a phosphate ester group.

12. An enzymatically cleavable 1,2-dioxetane compound as recited in claim 10 in which the enzymatically cleavable labile ring substituent is a galactoside group.

13. A 3-(2'"'"'-spiroadamantane)-4-methoxy-4-(6"-phosphoryloxy)naphth-1'"'"'-yl-1,2-dioxetane salt.

17. The disodium salt of a 1,2-dioxetane compound as in any one of claims 13-16, inclusive.

14. A 3-(2'"'"'-spiroadamantane)-4-methoxy-4-(7"-phosphoryloxy)naphth-2'"'"'-yl-1,2-dioxetane salt.

15. A 3-(2'"'"'-spiroadamantane)-4-methoxy-4-(8"-phosphoryloxy)naphth-1'"'"'-yl-1,2-dioxetane salt.

16. A 3-(2'"'"'-spiroadamantane)-4-methoxy-4-(5"-phosphoryloxy)naphth-2'"'"'-yl-1,2-dioxetane salt.

18. 3-(2,-spiroadamantane)-4-methoxy-4-(5"-β

-galactosyloxy)naphth-2'"'"'-yl-1,2-dioxetane.